Controlling the freezing process: a robotic device for rapidly freezing biological tissues with millisecond time resolution.

A robotic cryogenic device was developed which allows freezing of thick biological tissues with millisecond time resolution. The device consists of two horizontally oriented hammers (pre-cooled with liquid N(2)) driven by two linear servo-motors. The tissue sample is bathed in Ringers contained in a chamber which drops rapidly out of the way just as the hammers approach. A third linear motor is vertically oriented, and permits the rapidly dropping chamber to smoothly decelerate. All movements were performed by the three motors and four solenoids controlled by a PC. Mechanical adjustments, that change the size of the gap between the hammers at the end position, permit the final thickness of the frozen tissue to be varied. Here we show that the freezing time increased with the square of the final thickness of the frozen bundle. However, when bundles of different original thicknesses (up to at least 1mm) were compressed to the same final thickness (e.g., 0.2mm), they exhibited nearly equal freezing times. Hence, by being able to adjust the final thickness of the frozen bundles, the device not only speeds the rate of freezing, but standardizes the freezing time for different diameter samples. This permits the use of freezing for accurate determination of the kinetics of cellular processes in biological tissue.